Anti-Hsp90 alpha antibody

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp90 alpha antibody (AB2928)

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human kidney tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1 : 100 with a rabbit polyclonal antibody recognizing Heat Shock Protein 90 (86) ab2928 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp90 alpha antibody (AB2928)

ab2928 labelling Hsp90 alpha in Human colon adenocarcinoma tissue sections by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, heat-induced epitope retrieval was performed using 10mM sodium citrate (pH 6.0) buffer for 20 minutes at 95°C. Tissues were blocked in 3% BSA in PBST for 30 minutes at room temperature. Tissue sections were incubated with the primary antibody (1 : 100) for 1 hour. A HRP-conjugated goat anti-rabbit IgG (1 : 250) was used as the secondary antibody, followed by colorimetric detection using Metal Enhanced DAB Substrate Kit. Tissues were counterstained with hematoxylin and prepped for mouting. Images were taken at 40X magnification.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Hsp90 alpha antibody (AB2928)

Immunocytochemistry analysis of HeLa cells labeling HSP90 alpha with ab2928 at 5ug/mL in 0.1% BSA, incubated at 4°C overnight. Cells were 70% confluent log phase. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. Cells were then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 at 1/2000, for 45 minutes at room temperature (Panel a : Green). Nuclei (Panel b : Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI. F-actin (Panel c : Red) was stained with Rhodamine Phalloidin 1/300). Panel d represents the merged image showing cytoplasm and weak Nucleus localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Hsp90 alpha antibody (AB2928)

Immunocytochemistry/Immunofluorescence analysis of HSP90 alpha (green) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells and NIH/3T3 (Mouse embryo fibroblast cell line) cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were incubated with ab2928 at a dilution of 1 : 100 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488 goat-anti-rabbit IgG secondary antibody (1 : 400) for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp90 alpha antibody (AB2928)

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human breast carcinoma tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1 : 50 with a rabbit polyclonal antibody recognizing Heat Shock Protein 90 (86) ab2928 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp90 alpha antibody (AB2928)

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human tonsil tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1 : 200 with a rabbit polyclonal antibody recognizing Heat Shock Protein 90 (86) ab2928 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

• IP

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Immunoprecipitation - Anti-Hsp90 alpha antibody (AB2928)

Immunoprecipitation of HSP90 alpha was performed on HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. Antigen-antibody complexes formed by incubating 500ug whole cell lysate with 2ug of ab2928 overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Plus Agarose, washed extensively, and eluted with buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with ab2928 at a dilution of 1 : 1000 overnight rotating at 4°C. The membrane was washed in TBST, and probed with detection reagent at a dilution of 1 : 1000 for at least 1 hour. Chemiluminescent detection was performed.

All lanes:

Immunoprecipitation - Anti-Hsp90 alpha antibody (ab2928)

Predicted band size: 85 kDa

false

• WB

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Western blot - Anti-Hsp90 alpha antibody (AB2928)

Western blot analysis of HSP90 alpha was performed by loading samples onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with ab2928 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and probed with a secondary antibody for at least 1 hour. Chemiluminescent detection was performed.

All lanes:

Western blot - Anti-Hsp90 alpha antibody (ab2928) at 1/1000 dilution

Lane 1:

HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 50 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 50 µg

Lane 3:

K562 (Human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate at 50 µg

Lane 4:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 50 µg

Lane 5:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 50 µg

Lane 6:

COS-7 (African green monkey kidney fibroblast-like cell line) whole cell lysate at 50 µg

Lane 7:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate at 50 µg

Lane 8:

NRK (Rat kidney normal tissue) whole cell lysate at 50 µg

Secondary

All lanes:

Goat anti-rabbit IgG HRP secondary antibody at 1/20000 dilution

Predicted band size: 85 kDa

false

• WB

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Western blot - Anti-Hsp90 alpha antibody (AB2928)

Detected by chemiluminescence

All lanes:

Western blot - Anti-Hsp90 alpha antibody (ab2928) at 1/2000 dilution

Lane 1:

HeLa cell lysate at 30 µg

Lane 2:

A549 cell lysate at 30 µg

Lane 3:

LNCaP cell lysate at 30 µg

Lane 4:

NIH/3T3 cell lysate at 30 µg

Lane 5:

Mouse testis tissue lysate at 30 µg

Lane 6:

Rat testis tissue lysate at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant, HRP at 1/4000 dilution

Predicted band size: 85 kDa

Observed band size: 80 kDa

false